Isoprene (2-methyl-1,3-butadiene) is a volatile hydrocarbon that is insoluble in water and soluble in alcohol. Commercially viable quantities of isoprene can be obtained by direct isolation from petroleum C5 cracking fractions or by dehydration of C5 isoalkanes or isoalkenes (Weissermel and Arpe, Industrial Organic Chemistry, 4th ed., Wiley-VCH, pp. 117-122, 2003). The C5 skeleton can also be synthesized from smaller subunits. It would be desirable, however, to have a commercially viable method of producing isoprene that was independent of nonrenewable resources.
Isoprene monomer is employed in the manufacture of polyisoprene and various copolymers (with isobutylene, butadiene, styrene, or other monomers). Building a strain (prokaryotic or eukaryotic) capable of producing commercially viable levels of isoprene requires optimization of part of or the entire DXP or MVA pathway or both MVA and DXP pathways. A key enzyme in the pathway is isoprene synthase (IspS), which converts the precursor DMAPP to isoprene. Isoprene synthases (IspS) that have been identified include those from plants such as poplar, English oak and kudzu vine. Some of the identified plant IspS enzymes have been partially characterized in part by expression in E. coli and some of the kinetic parameters of these enzymes have been determined in vitro with purified protein. However, kinetic parameters, including Km, Ki, and Kcat, of the native IspS enzymes and even some of the recombinant enzymes are insufficient for commercial production of isoprene in a biological host. In addition, commercial use of some IspS enzymes can be limited due to insufficient expression levels. Thus, one problem to be solved is the provision of isoprene synthases that have improved properties such that a greater amount of isoprene can be biologically produced for commercial use. To solve this problem as described herein, an isoprene synthase with improved properties may be expressed in a host (e.g. a bacterial host). In particular, the present invention provides legume isoprene synthases that exhibit improved properties for increased isoprene production in host cells.
All patents, patent applications, publications, documents, nucleotide and protein sequence database accession numbers, the sequences to which they refer, and articles cited herein are all incorporated herein by reference in their entireties.